Regulation of oxygen metabolism in human granulocytes: Relationship between stimulus binding and oxidative response using plant lectins as probes

Abstract

To examine the relationship between binding of surface-active stimuli to human neutrophils and activation of the superoxide (O2-) and peroxide (H2O2) forming oxidase(s) of these cells, oxygen (O2) consumption and O2- and H2O2 release were measured following stimulation by plant lectins. Both concanavalin A (Con-A) and phytohemagglutinin (PHA) were more active than other lectins in stimulating the respiratory activity of neutrophils. When the cells were pretreated with cytochalasin B (Cyto-B), recovery of extracellular O2- and H2O2 was increased by 3-10-fold, as found previously with particulate stimuli. Whereas cytochalasin B reduced O2 consumption by cells triggered with particulate stimuli, cytochalasin B treatment of cells produced more than a threefold increase in O2 consumption by cells triggered with plant lectins. The stimulatory effect of cytochalasin B on the respiration of Con-A- or PHA-treated cells was rapid, with an enhancement observed within 60 sec. With Con-A-stimulated cells, it could be inhibited by alpha-methyl-D-mannoside (AMM), indicating that it was specifically due to an activity mediated by the lectin as opposed to some other action of cytochalasin B. To correlate these events with binding of the lectin to the cells, 3H-Con-A was employed. Pretreatment of cells with Cyto-B increased 3H-Con-A binding in direct parallel to the stimulation of respiration. Similar results were found when the Cyto-B was added in the middle of an incubation period. Both the increased binding and respiratory activity of the cytochalasin-B-treated cells could be inhibited by the simultaneous addition of AMM. These findings support the concept that the degree of activation of the neutrophil oxidase(s) is a direct function of the extent of binding of the initial stimulus. They also suggest that on-going stimulus binding is a prerequisite for continued respiratory activity. The precise nature of the lectin binding sites 'uncovered' in Cyto-B-treated cells and their potential relationship to sites of oxidase activity or regulation remains to be determined.